S151: HIGH-THROUGHPUT EVALUATION OF THE POTENTIAL OF CANCER DRUGS TO ENHANCE NATURAL KILLER CELL IMMUNOTHERAPY IN CHRONIC MYELOID LEUKEMIA.
Authors: P. Nygren, J. Bouhlal, E. Laajala, A. Ianevski, J. Klievink, H. Lähteenmäki, K. Saeed, D. Lee, T. Aittokallio, O. Dufva, S. Mustjoki
Journal: HemaSphere (2022)

Background: Natural killer (NK) cell immunotherapies are promising novel cancer treatments and complete remission has been achieved in patients with relapsed/refractory myeloid leukemias. The significance of NK cells in chronic myeloid leukemia (CML) patients has been highlighted in several studies. CML patients with a higher percentage of mature NK cells have increased relapse-free survival after treatment discontinuation. NK KIR receptor profiles have also been associated with the achievement of remission in response to tyrosine kinase inhibitor (TKI) therapy. Moreover, complex interactions between TKIs and NK cell function have been discovered in CML. For example, dasatinib and imatinib have been suggested to enhance NK cell cytotoxicity through regulation of activating and inhibitory receptors. NK cell responses are usually short-lived and attempts have been made to enhance their function against malignant cells using cytokines, bi- and tri-specific antibodies, and small molecule inhibitors. However, a large-scale drug screening evaluating the combinatorial effects of NK cells and cancer drugs has not been previously conducted. Aims: To evaluate the potential of small molecule cancer drugs to synergize with NK cell immunotherapy in CML and to discover which drugs can enhance or inhibit cytotoxicity in CML cells. Methods: A high-throughput drug sensitivity and resistance testing (DSRT) screen was used to evaluate the effect of 528 investigational and approved cancer drugs on the cytotoxicity of NK cells. CML cells (K562) expressing luciferase and primary expanded NK cells were co-cultured for 24 hours on 384 well drug plates. Target cell viability was measured using a luciferase-based readout, and results were run through a custom pipeline to calculate differential drug sensitivity scores (dDSS) between co-cultured cells and target cells alone, for each drug. Most promising drugs with highest and lowest dDSS were selected for further analysis with single-cell RNA sequencing to determine their mechanism of action. Results: Out of 528 screened drugs, 161 had an inhibitory effect on cytotoxicity (dDSS < -2.5), 325 had no effect, and 42 had an enhancing effect (dDSS > 2.5). The SMAC mimetics birinapant and LCL-161 were the most potent enhancers of NK cell cytotoxicity and had no effect on target cells alone. Individual drugs with other mechanisms, such as PKC activators, were also effective enhancers of NK cytotoxicity. On the contrary, cytotoxicity-inhibiting drugs included previously known immunosuppressive drugs dexamethasone and prednisolone, as well as novel candidates, such as sotrastaurin, a PKC inhibitor. Single-cell RNA sequencing of drug-treated co-cultures revealed the transcriptomic phenotypes of NK cell and target cells under SMAC mimetic treatment. NF-kB target genes such as BIRC3, NFKB2, MHC II genes, and the T/NK cell-recruiting chemokines CXCL9, CXCL10, and CXCL11 were activated in target cells exposed to both birinapant and NK cells, compared to target cells exposed to NK cells alone (p value adj < 0.05). These data suggest that SMAC mimetics in combination with NK cells may induce an immune-inflamed phenotype in addition to sensitizing CML cells to NK cell cytotoxicity. Image:Summary/Conclusion: We discovered novel drug classes, which had both activating and inhibitory effects on the NK cell cytotoxicity against CML cells in vitro. Defining NK cell - drug - CML cell interactions in a high-throughput setting provides a framework for future combination immunotherapies to further improve treatment-free remission in CML.